It is known that, as a consequence of various biochemical processes in biological systems (e.g. redox processes in the respiratory chain, oxidations in the cytoplasm), O.sub.2 radicals are continuously formed which are highly cytotoxic and which may lead to tissue damage. In pathological situations, e.g. in the course of rheumatic disorders, it is believed that degradation of collagen and synovial fluid is caused by such radicals (Pasquier, C. et al., Inflammation 8:27-32, (1984)).
Eukaryotic cells contain primarily two forms of superoxide dismutases (SOD), one of which occurs predominantly in the cytosol (Cu/Zn-SOD) and the other in the mitochondria (Mn-SOD). In liver mitochondria, it has been found that the Mn-enzyme is located in the matrix enclosing the inner membrane. Mn-SOD has also been detected in the cytosol of liver cells (McCord J. M. et al. In: Superoxide and Superoxide Dismutases (A. M. Michelson, J. M. McCord, I. Fridovich, eds.) Academic Press, N.Y., 129-138, (1977)).
In prokaryotes, there is an Fe-SOD in addition to a MnSOD. Fe-SOD has also been detected in algae and protozoa and in some plant species (Bridges, S. M., Salin, M. L., Plant Physiol. 68:275-278 (1981)). These highly active enzymes catalyze the disportionation O.sub.2 +O.sub.2 +2H.sup.+ .fwdarw.H.sub.2 O.sub.2 +O.sub.2. By this dismutation of the superoxide radicals, the concentration of the radicals and hence cell damage is prevented. Apart from the endoplasmic reticulum of the liver, the mitochondrial membranes are regarded as one of the most important sites of O.sub.2 production in animal cells. Thus, it is not surprising that mitochondria have their own special SOD (Mn-SOD).
The structural gene of a prokaryotic Mn-SOD (E. coli) has been cloned and the chromosomal sodA-gene has been located (Touati, D., J. Bact. 155: 1078-1087 (1983)).
The 699 bp long nucleotide sequence of a mitochondrial yeast Mn-SOD has been clarified and the primary structure both Of the precursor and of the mature protein have been derived therefrom--with molecular weights of 26123 Da for the precursor and 23059 Da for the mature. protein (Marres, C. A. M. et al., Eur. J. Biochem. 147:153-161 (1985)). Thus, the Mn and Cu/ZnSOD (MW=14893, EP-A-138111) differ significantly in their molecular weights.
The complete amino acid sequence of Mn-SOD from human liver has been published by Barra D., et al. J. Biol. Chem. 259:12595-12601 (1984). According to Barra et al., hMn-SOD consists of 196 amino acids. Human Cu/Zn-SOD from erythrocytes, on the other hand, consists of 153 amino acids (Jabusch, J. R., et. al. Biochemistry 19:2310-2316, (1980) and exhibits no sequence homologies to hMn-SOD (Barra, D. et al., see above).
The preparation of the SOD, particularly by the methods of DNA recombination, is well known in the art and has been frequently described. For example, the human Cu/Zn-SOD may be prepared according to EP-A 0 138 111, EP-A 0 180 964, WO 85/01503 and WO 91/06634. Human Mn-SOD can be obtained using the method described in EP-A 0 282 899. The preparation of EC-SOD is described in EP-A 0 236 385 and EP-A 0 492 447. Fe-SOD may be prepared according to EP-A 218 480 and vegetable SODs may be prepared according to EP-A 0 359 617, EP-A 0 356 061 and WO 90/01260. An Mn-SOD frown Serratia is disclosed in EP-A 0 210 761.
The preparation and recovery of SOD by Conventional methods, e.g. by extraction from cells and/or tissues of animal or human origin and subsequent purification, e.g. by chromatographic separation, has also been described elsewhere (e.g. EP-A 0 188 053, DE-OS 3124 228). In addition, chemically modified derivatives of SOD or SOD-analogs are well known and are disclosed for example in EP-A 0 292 321, EPA 0 483 113 and WO 89/012677.
Numerous attempts have been made to stabilize biologically active proteins by the addition of various substances and mixtures. For example, it is proposed according to EP-A 0 142 345 to stabilize interferons by adding a protease inhibitor; alternatively, sugar alcohols are described for achieving a stabilizing effect (EPA 0 080 879). In addition to these stabilizers, human serum albumin (EP-A 0 162 332), dextrans, dextrins and other saccharides (EP-A 0 123 291), albumin with sugar or sugar alcohols (EPA 0 231 132), gelatine (EP-A 0 091 258) or certain buffer systems (WO 88/09674) have also been disclosed as stabilizing agents in biologically active proteins (interferons, TNF, IL-2).
WO 90/03784 describes the use of .beta.- and .gamma.-cyclodextrin derivatives, particularly hydroxypropyl-.beta.-cyclodextrin (HPBCD) for stabilizing various biologically active proteins. This patent application describes stabilization or solubilization or the prevention of aggregation with HPBCD, in particular, explicitly for interleukin-2 (IL-2), tumor necrosis factor (TNF), macrophage colony stimulating factor (m-CSF), insulin and human growth hormone (HGH). See also, Brewster, M. E. et al., Pharm. Res 8:792-795 (1991).
It is known from Lee, M. and Fennema O. R., J. Agric. Food Chem. 39:17-21 (1991), that cyclodextrins prevent the clumping of .beta.-caseine.
The publication by Hora M. S. et al., Pharm. Res. 9:33-36 (1992), discloses how the formation of soluble dimers of tumor necrosis factor (TNF) in lyophilized form can be prevented if before the lyophilization, mannitol is added to the TNF together with an amorphous component (dextran, saccharose, trehalose or 2- hydroxypropyl-.beta.-cyclodextrin).